Starch Granule Initiation in Arabidopsis Requires the Presence of Either Class IV or Class III Starch Synthases

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

Starch granule initiation in Arabidopsis thaliana chloroplasts

The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts

A novel mutant of Arabidopsis thaliana, arc6 (accumulation and replication of chloroplasts), has been isolated from a transfer DNA-mutagenized population of Arabidopsis seedlings. arc6 has the most extreme arc mutant phenotype we have yet described, with only one to three chloroplasts per leaf mesophyll cell compared to a mean of 83 in cells of the wild-type var Wassilewskija. The chloroplasts of arc6 are 20-fold larger than wild-type chloroplasts.Chloroplast division is almost certainly precluded in arc6 mesophyll cells, since chloroplast number per cell does not increase during mesophyll cell expansion. arc6 chloroplasts are long and thin in cross-section and only one-half the width of wild-type chloroplasts and the arrangement of thylakoid membranes is largely unaltered. arc6 segregates as a monogenic recessive nuclear mutation in a normal Mendelian manner and the arc6 phenotype is stably inherited for at least four generations. arc6 plants grow normally and are fertile, although the rosette leaves appear curled and twisted. arc6 plants accumulate 70 to 75% of the biomass of wild type. The phenotype of this novel mutant is discussed in relation to the nature of the control of chloroplast division in leaf cells.     

小麦黄化突变体叶绿体超微结构研究

利用透射电镜对小麦自然黄化突变体及其突变亲本(西农1718)叶片细胞叶绿体的数目、形态及超微结构进行比较分析。结果发现:(1)3种不同黄化程度突变体的叶绿体分布、数目、形状及大小与突变亲本无明显差异;(2)突变体叶绿素含量为野生型58%的黄绿植株与其突变亲本叶绿体超微结构无明显差异,基质类囊体与基粒类囊体高度分化,基粒数目以及基粒片层数目较多;(3)突变体金黄和绿黄植株的叶绿素含量分别为野生型的17%、24%,其叶绿体超微结构与突变亲本明显不同,突变体的叶绿体发育存在明显缺陷,其中突变体金黄植株的叶绿体内无基粒、基质片层清晰可见,有淀粉粒,嗜锇颗粒较多,而突变体绿黄植株的叶绿体内有基粒,但明显少于突变亲本,且基粒片层较少,基质类囊体较发达。结果表明该黄化突变体叶绿体超微结构的改变,是由于叶绿素含量降低造成,推测,该黄化突变是由于叶绿素合成受阻导致的。     

Morphometric Analysis of Chloroplasts of Cotton Leaf and Fruiting Organs

We examined morphological and ultrastructural differences in chloroplasts of cotton leaves and the fruiting organs, bract, and capsule wall to advance our understanding of their commonly observed differences in photosynthetic efficiency. Chloroplasts from leaves were large (7.1 μm long in cross section), lens shaped with a well developed membrane system differentiated into grana and stroma lamellae that occupied the large cross-sectional area (12.3 μm²) of the organelle. A few small plastoglobuli and starch grains were scattered in the stroma region. The bract chloroplasts were correlative of leaf chloroplasts in size (6.8 μm in length) and shape with the exception that the bract chloroplasts exhibited greater thylakoid number per granum (15.8) than the leaf chloroplasts (10.5). In contrast to leaf and bract, the capsule wall chloroplasts were smaller in size (4.3 μm) and cross sectional area (6.8 μm²) than either the leaf or bract. The most intriguing feature of the capsule wall chloroplasts was its domination by large starch granules (5.3 μm²) in the stroma which filled the whole chloroplast coercing the membrane system to move towards the periphery of the organelle. Grana number and thylakoids per granum were lowest in the capsule wall chloroplasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Arabidopsis thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light

The effects of chloroplast number and size on the capacity for blue light-dependent chloroplast movement, the ability to increase light absorption under low light, and the susceptibility to photoinhibition were investigated in Arabidopsis thaliana. Leaves of wild-type and chloroplast number mutants with mean chloroplast numbers ranging from 120 to two per mesophyll cell were analysed. Chloroplast movement was monitored as changes in light transmission through the leaves. Light transmission was used as an indicator of the ability of leaves to optimize light absorption. The ability of leaves to deal with 3 h of high light stress at 10 degrees C and their capacity to recover in low light was determined by measuring photochemical efficiencies of PSII using chlorophyll a fluorescence. Chloroplast movement was comparable in leaves ranging in chloroplast numbers from 120 to 30 per mesophyll cell: the final light transmission levels after exposure to 0.1 (accumulation response) and 100 micromol photons m(-2) s(-1) (avoidance response) were indistinguishable, the chloroplasts responded quickly to small increases in light intensity and the kinetics of movement were similar. However, when chloroplast numbers per mesophyll cell decreased to 18 or below, the accumulation response was significantly reduced. The avoidance response was only impaired in mutants with nine or fewer chloroplasts, both in terms of final transmission levels and the speed of movement. Only mutants lacking both blue light receptors (phot1/phot2) or those with drastically reduced chloroplast numbers and severely impacted avoidance responses showed a reduced ability to recover from high light stress.     

The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation

All plants and green algae synthesize starch through the action of the same five classes of elongation enzymes: the starch synthases. Arabidopsis mutants defective for the synthesis of the soluble starch synthase IV (SSIV) type of elongation enzyme have now been characterized. The mutant plants displayed a severe growth defect but nonetheless accumulated near to normal levels of polysaccharide storage. Detailed structural analysis has failed to yield any change in starch granule structure. However, the number of granules per plastid has dramatically decreased leading to a large increase in their size. These results, which distinguish the SSIV mutants from all other mutants reported to date, suggest a specific function of this enzyme class in the control of granule numbers. We speculate therefore that SSIV could be selectively involved in the priming of starch granule formation.     

Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.     

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.     

The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence

Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.     

墨兰幼叶和成熟叶不同部位叶绿体超微结构和光合作用

墨兰试管苗植株成熟叶片叶绿体基粒较发达,类囊本膜垛叠较紧密。幼叶叶绿体中少有亲锇颗粒,成熟叶的叶绿体中往往既有亲锇颗粒又有淀粉粒。幼叶中基粒数目比成熟叶的少,叶绿体也比成熟叶的小。幼叶的光合放氧速率比成熟叶的低。幼叶中叶尖部叶绿体最大而叶基部最小,但叶尖部的光合放氧速率比叶基部小。成熟叶中叶绿体大小及光合放氧速率区别不明显。通过对各部位叶绿素含量的测定发现,叶绿素含量与光合放氧速率之间没有正相关性     

Chloroplast-ultrastructure and chlorophyll content in leaves from Quercus branches with and without epiphytic lichen thalli

Abstract The effect that the massive presence of lichen thalli growing on the branches of Quercus pyrenaica and Q. rotundifolia leaves has on their chloroplasts been studied. In both species there were significant decreases in the amount of chlorophylls in the leaves of twigs with a dense cover of lichens in comparison with the leaves from thallus-free twigs. The areas and perimeter of chloroplasts in leaves from twigs with epiphytes did not differ significantly from those in leaves without epiphytes. However, in leaves with epiphytes the percentage of chloroplast area occupied by starch was higher. In Q. pyrenaica the number of grana per chloroplast section and per μm², the percentage of chloroplast stroma occupied by grana, the average number of thylakoids forming grana and the grana width was significantly smaller in leaves near lichen populations. These results are discussed and related to the great chelating capacity of the lichen's substances.     

Changes in the plastid ultrastructure duringSedum rotundifolium leaf development

The ultrastructure of plastids was investigated in succulent leaves ofSedum rotundifolium to examine their changes during development. Leaves were categorized as etiolated, immature, young, and mature, based on their developmental stage and size. Of particular interest were the features of the tubular inclusion bodies (TIBs) and starch grains. These, along with vacuole size, showed remarkable changes over time. Etioplasts of unexposed leaves had prolamellar bodies, abundant starch grains, large TIB, few plastoglobuli, and thylakoid systems. Membranes of the thylakoids were still continuous with those of the prolamellar body. The plastids were often influenced by the presence and profile of inclusion bodies and starch grains throughout the early stages. Morphology was highly variable in the etioplasts but consistently hemispherical or ovoid in mature chloroplasts. TIB was most abundant in the etiolated leaves, but disappeared completely with development. Starch grains also became significantly reduced in size. Both young and mature mesophyll cells exhibited a normal chloroplast ultra-structure and huge central vacuoles, with an extremely thin peripheral cytoplasm. Grana were extensive and comprised a large portion of the chloroplasts. Traces of peripheral reticulum were also discovered in the chloroplasts of expanded leaves. The implications of these ultrastructural changes in the tubular inclusions and starch grains are discussed with relevance to Crassulacean acid metabolism (CAM).     

Evidence for distinct mechanisms of starch granule breakdown in plants

The aim of this work was to understand the initial steps of starch breakdown inside chloroplasts. In the non-living endosperm of germinating cereal grains, starch breakdown is initiated by alpha-amylase secreted from surrounding cells. However, loss of alpha-amylase from Arabidopsis does not prevent chloroplastic starch breakdown (Yu, T.-S., Zeeman, S. C., Thorneycroft, D., Fulton, D. C., Dunstan, H., Lue, W.-L., Hegemann, B., Tung, S.-Y., Umemoto, T., Chapple, A., Tsai, D.-L., Wang, S.-M, Smith, A. M., Chen, J., and Smith, S. M. (2005) J. Biol. Chem. 280, 9773-9779), implying that other enzymes must attack the starch granule. Here, we present evidence that the debranching enzyme isoamylase 3 (ISA3) acts at the surface of the starch granule. Atisa3 mutants have more leaf starch and a slower rate of starch breakdown than wild-type plants. The amylopectin of Atisa3 contains many very short branches and ISA3-GFP localizes to granule-like structures inside chloroplasts. We suggest that ISA3 removes short branches from the granule surface. To understand how some starch is still degraded in Atisa3 mutants we eliminated a second debranching enzyme, limit dextrinase (pullulanase-type). Atlda mutants are indistinguishable from the wild type. However, the Atisa3/Atlda double mutant has a more severe starch-excess phenotype and a slower rate of starch breakdown than Atisa3 single mutants. The double mutant accumulates soluble branched oligosaccharides (limit dextrins) that are undetectable in the wild-type and the single mutants. Together these results suggest that glucan debranching occurs primarily at the granule surface via ISA3, but in its absence soluble branched glucans are debranched in the stroma via limit dextrinase. Consistent with this model, chloroplastic alpha-amylase AtAMY3, which could release soluble branched glucans, is induced in Atisa3 and in the Atisa3/Atlda double mutant.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.